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Figure 1. Effects of infection with attenuated knockout (KO) FV3 recombinant or bacterial stimulation on iVα6 T cell response. Peritoneal leukocytes (PLs) were collected at 1 dpi from adult frogs infected with 1 à 106 PFUs of WT-FV3 or â64-FV3, or 100 µl heat-killed (HB) E. coli. (A) Genome copy number using absolute qPCR with primers against FV3 DNA polymerase II. (B) XNC10 relative gene expression and (C) iVα6-Jα1.43 relative gene expression. Gene expression was determined relative to an endogenous control (GAPDH) and fold changes were calculated using the unstimulated sample (injected with equivalent volume of APBS) collected at the same time point. Data are pooled from three independent experiments with n = 4â5 animals in each experiment and each dot represents an individual animal. The line intersecting the y-axis at 0 represents the unstimulated control that the fold changes of the treatments are in relation to; (#) p < 0.05 significant differences compared to unchallenged (APBS) injected controls; (*) p < 0.05 and (***) p < 0.001 statistically significant differences between the indicated groups (one way ANOVA and Dunnsâs multiple comparison test).
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Figure 2. Magnitude of iVα6 T cell response in adult frogs is associated to the level of viral replication and production of infectious particles. PLs and kidneys were collected at 1, 3, and 6 dpi from adult frogs infected with 1 à 106 PFUs of WT-or â64-FV3. FV3 genome copy number by absolute qPCR in PLs (A) and kidneys (B) were determined. The total number of infectious particles (nd: not detected) in the kidney was determined by plaque assay (C).Gene expression of iVα6-Jα1.43 and XNC10 in PLs (D,F) and kidneys (E,G) were determined relative to an endogenous control (GAPDH) and fold changes were calculated using mock-infected frogs as a control. Each dot represents an individual animal (n = 4â5). The line intersecting the y-axis at 0 represents the APBS control that the fold changes of the treatments are in relation to. Note: * p < 0,05; ** p < 0.01; *** p < 0.001, and **** p < 0.0001 above the line denotes statistically significant differences between the different treatment groups; significant differences between time points within each treatment group are indicated within parentheses; NS indicates no significant differences (one way ANOVA and Dunnsâs multiple comparison test).
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Figure 3. The iVα6 T cell response in tadpoles depends on active viral replication and productive FV3 infection. Three week-old (stage 55) tadpoles were infected with 10,000 PFUs of WT-or â64-FV3. At 1, 3, and 6 dpi, kidneys were collected and the total number of infectious particles was determined by plaque assay, respectively (A). Gene expression of iVα6-Jα1.43 (B) and XNC10 (C) was determined relative to an endogenous control (GAPDH) and relative expression was calculated against the lowest observed expression according to the ââCt method (n = 5). No iVα6-Jα1.43 transcripts were detected in the kidney uninfected tadpoles. * p < 0.05 and ** p < 0.01 above the line denotes statistically significant differences between treatment groups; NS indicates no significant differences (one way ANOVA and Dunnâs multiple comparison test).
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Figure 4. XNC10 tetramer-mediated iVα6T cell depletion in tadpoles affects iVα6-Jα1.43 transcript levels and viral replication. PLs and kidneys were collected from three week-old (stage 55) tadpoles that had been injected with 1 μg XNC10 tetramers (XNC10-T) or vehicle control 1 day pre, and 1 day post i.p. injected with 10,000 PFUs of FV3, at the indicated time points (n = 9). A schematic of the injection regime is shown in (A). Gene expression of iVα6-Jα1.43 in PLs (B) and kidneys (C) is shown. Results are normalized to an endogenous control and presented as relative expression compared with the lowest observed value according to the ∆∆Ct method. FV3 loads in PLs (D) and kidneys (E) were measured using absolute qPCR with primers against FV3 polymerase II. For PLs, each dot represents a pool of 3 tadpoles, while for kidneys, each dot represents a single tadpole; * p < 0.05 denotes statistically significant differences between the indicated groups; NS indicates no significant differences (One way ANOVA followed by Tukey’s multiple comparison test).
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Figure 5. Specificity and long term impact of transitory iVα6T cell depletion. (A) Three week-old (stage 55) tadpoles were injected with either 1 μg XNC10-tetramers (FV3/XNC10-T), 1 μg XNC10-monomers (FV3/XNC10-M), or vehicle control (FV3/APBS) 1 day pre and 1 day post i.p. infection with 10,000 PFUs of FV3 (n = 8), and kidneys were collected at 6 dpi. The last group (FV3/XNC10-T priming) was first injected with 1 μg XNC10-tetramers, then 3 days later infected with FV3, and kidneys were collected at 6 dpi. Viral loads were assessed by absolute qPCR with primers against FV3 polymerase II. The results are combined from two separate experiments, and each dot represents an individual tadpole; ** p < 0.01 above the line denotes statistically significant differences between the indicated groups (One way ANOVA followed by Tukey’s multiple comparison test). (B) Three week-old (stage 55) tadpoles were injected with either 1 μg XNC10 tetramers (FV3/XNC10-T) or vehicle control (FV3/APBS) 1 day pre, and 1 day post were i.p injected with 10,000 PFUs of FV3 (n = 15) and survival was monitored daily over a 30-day period. Survival was determined using Kaplan-Meier, * p < 0.05, ** p < 0.005, and *** p < 0.0005. Uninfected controls (white circle), FV3 infected tadpoles (black circle), and XNC10 tetramer treated FV3 infected tadpoles (grey circle).
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Figure 6. Effect of XNC10 tetramer treatment on the expression of the macrophage stimulating factor genes CSF-1 and IL-34 in the peritoneal cavity and kidneys of FV3 infected tadpoles. PLs and kidneys were collected from three week-old (stage 55) tadpoles that had been injected with 1 μg XNC10 tetramers or vehicle control 1 day pre- and 1 day post-i.p. injection with 10,000 PFUs of FV3, at the indicated time points (n = 8–9). Quantitative gene expression analysis of IL-34 (A,B) and CSF-1(C,D) were determined relative to a endogenous control (GAPDH), and relative expression was calculated against the lowest observed expression according to the ∆∆Ct method (n = 9); ** p < 0.005 above the line denotes statistically significant differences between the different treatment groups; NS indicates no significant differences (One way ANOVA followed by Tukey’s multiple comparison test).
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Figure 7. XNC10 tetramer treatment results in a delayed antiviral response in the peritoneal cavity and kidneys of FV3 infected tadpoles. PLs and kidneys were collected from three week-old (stage 55) tadpoles that had been injected with 1 μg XNC10 tetramers (XNC10-T) or vehicle control 1 day pre- and 1 day post-i.p. injection with 10,000 PFUs of FV3, at the indicated time points (n = 8â9). Quantitative gene expression analysis of type I (A,D), type II (B,E), and type III IFN (C,F) were determined relative to a endogenous control (GAPDH), and relative expression was calculated against the lowest observed expression according to the ââCt method (n = 9); * p < 0.05, ** p < 0.005, and *** p < 0.001 above the line denote statistically significant differences between the different treatment groups; significant differences between time points within each treatment group is indicated within parentheses; NS indicates no significant differences (One way ANOVA followed by Tukeyâs multiple comparison test).
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Figure 8. Effects of iVα6T cell depletion on IL-18 and IL-12 responses. PLs and kidneys were collected from three week-old (stage 55) tadpoles that had been injected with 1 μg XNC10 tetramers (XNC10-T) or vehicle control 1 day pre- and 1 day post-i.p. injection with 10,000 PFUs of FV3, at the indicated time points (n = 8â9). Quantitative gene expression of IL-18 (A,B) and type IL-12 (C,D) was determined relative to an endogenous control (GAPDH) and relative expression was calculated against the lowest observed expression according to the ââCt method (n = 9); * p < 0.05; ** p < 0.005, above the line denotes statistically significant differences between the different treatment groups; significant differences between time points within each treatment group is indicated within parentheses; NS indicates no significant differences (One way ANOVA followed by Tukeyâs multiple comparisons test).
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